XBP1在电针减轻心肌缺血再灌注损伤中的作用及其分子机制

Acupuncture is the treasure of Chinese medicine. Electroacupuncture preconditioning (EA) has been shown to attenuate myocardial ischemia reperfusion injury. The early research of the applicant and research team found that the protective effect of EA was associated with endogenous opioid peptide system and cannabinoid system. In the whole process, endoplasmic reticulum stress mediated autophagy plays an important role, and the mechanism of the key molecule of XBP1 has not been clearly explained. The applicant has long been engaged in basic research on the endogenous protective mechanism among EA preconditioning, endoplasmic reticulum stress and autophagy, etc, and has published 15 SCI articles as the first or corresponding author. The results of the pre-experiment of the key molecules of XBP1 showed that EA pretreatment could increase the expression of XBP1 protein, and significantly reduce the apoptosis of myocardial cells after myocardial ischemia reperfusion injury, indicating that the protective effect of electroacupuncture may be related to the endogenous autophagy of XBP1. This project intends to use XBP1 key molecules as the breakthrough point. By comprehensively using gene knockout animals, GFP-LC3 fusion protein indication technology and immunofluorescence microscopy, this project aims to confirm that EA pretreatment could promote the aggregation of Beclin1 on the double layer of mitochondrial membrane, restore the level of autophagy in myocardial cells, and play an important role of myocardial protection. Meanwhile, the purpose of this project is to provide a new theoretical basis for the treatment of myocardial ischemia reperfusion injury and the regulation of myocardial cell environment of EA.

电针是祖国医学的瑰宝。电针预处理（EA）可以减轻心肌缺血再灌注损伤已被证实。申请者团队前期的研究发现EA的保护作用与内源性阿片肽系统及大麻素系统有关，其中内质网应激介导的自噬发挥了重要的作用，而关键分子XBP1的作用机制尚不清楚。申请者长期从事电针、内质网应激与自噬等内源性保护机制的研究，以第一或通讯作者发表相关SCI论文15篇。针对内质网应激关键分子XBP1的预实验显示，EA显著减轻缺血再灌注后心肌细胞的凋亡且XBP1蛋白表达明显升高，自噬双层膜结构也更为完整，提示EA的保护作用可能与XBP1启动心肌内源性自噬有关。本课题拟以XBP1为切入点，应用转基因大鼠、GFP-LC3示踪等技术，证实EA可能通过XBP1，促进Beclin1在双层膜上的聚集，保证自噬双层膜结构的完整性，恢复心肌细胞自噬水平，发挥损伤心肌的保护作用。为EA治疗缺血再灌注损伤心肌，整体调控心肌细胞内环境提供新的理论依据。

最新的统计结果显示2021年我国开展各类手术约7000万例。病人在手术中因基础疾病(如心、脑血管疾病)应激、出血、手术操作(体外循环、血管阻闭等)以及麻醉药物毒副作用等，可导致重要脏器的缺血再灌注损伤。心肌缺血/再灌注损伤(myocardial ischemia/reperfusion, MI/R)的问题在临床上十分常见，当出现MI/R时可导致严重的心功能不全。以往研究已证明电针(Electroacupunture，EA)对MI/R具有保护作用。.众所周知，自噬(autophagy)是损伤心肌重要的内源性保护机制。申请者既往的研究发现，当心肌发生损伤后，通过调节自噬，可以抑制心肌细胞凋亡，减轻过度的内质网应激，参与心肌细胞的修复。内质网应激重要信号分子X盒结合蛋白1(X-box binding protein 1, XBP1)是调控自噬的关键信号分子，该分子在EA保护中的作用机制目前尚不清楚。因此，我们设计此项研究证明EA可显著改善心MI/R期间的心功能，并减少心肌梗死面积，研究EA对MI/R的保护作用以及XBP1/GRP78信号在这一过程中的潜在作用。这些发现可能为临床应用EA预防MI/R损伤提供理论依据。.本研究发现，在功能学部分，EA可以明显改善MI/R后24小时后LVEF和LVFS的值，改善心肌缺血再灌注损伤后24小时后梗死面积容积。在具体的机制研究中，我们发现，EA通过降低血清乳酸脱氢酶（LDH）和肌酸激酶-MB（CK-MB）活性进一步抑制心肌损伤。结果还表明，EA增加了XBP1、葡萄糖调节蛋白78（GRP78）、Akt和Bcl-2的表达，并降低Bax和裂解的Caspase3的表达。通过体外使用XBP1抑制剂，结果表明，抑制XBP1表达可显著增加LDH和CK-MB的活性和细胞凋亡，从而加剧离体模拟的缺血/再灌注诱导的H9c2细胞损伤。与离体模拟的缺血/再灌注组相比，XBP1抑制离体模拟的缺血/再灌注损伤时下游GRP78和Akt的表达。总之，我们的研究表明，EA激活XBP1/GRP78/Akt信号通路，保护心脏免受心肌缺血/再灌注损伤。.这些发现揭示了电针抗MI/R的内在机制，证实 XBP1 是心肌缺血再灌注损伤细胞自噬的新靶点，为EA治疗MI/R心肌，整体调控心肌细胞内环境提供新的理论依据。